An investigation of Cytochrome P450 function in human cell Lines for potential use in a bioartificial liver device.
Doctoral thesis, UCL (University College London).
Bioartificial Liver Devices (BAL) could provide hepatic function in patients with liver failure awaiting transplantation or liver regeneration. Certain BALs use human hepatoblastoma-derived HepG2 cells that can perform many hepatocyte specific functions, however they cannot adequately detoxify xenobiotics via Cytochrome P450 (CYP) mediated pathways. The aim of this thesis was to identify and develop a cell source able to provide CYP function in a BAL, and focused on CYP1A2, since its inducible function has been well established in HepG2 cells and CYP3A due its large contribution to hepatic CYP expression and function. Firstly, gold standard comparators of freshly isolated primary human hepatocytes (PHH) were established. The influence of BAL culture conditions on CYP function were evaluated, including chemical induction, culture media composition, alginate encapsulation and microgravity 3D culture: HepG2 cells were able to provide sufficient CYP1A2 but limited CYP3A function. Intestinal cell lines LS147T and Caco2 and the hepatocyte derived cell line HC-04 were investigated as potential sources to provide CYP3A BAL function. All three were shown to proliferate in BAL specific medium within a 3D culture system. Under these conditions, increased CYP3A function was demonstrated for each cell line, but their usefulness within our BAL was limited. The effects of increasing HepG2 expression of two nuclear receptors (Constitutive Androstane Receptor and Retinoid X Receptor α) were examined. Within a transient system, increased nuclear receptor expression, in conjunction with chemical induction, resulted in improved function of multiple CYP isoforms; CYP3A function was increased to levels approaching those measured in PHH. Inducible CYP3A function of a stable CAR transfectant was then demonstrated in 3D culture. Lastly HepG2 cell CYP function was also shown to be source-dependent. Overall it was concluded that through adaptation of culture conditions and genetic manipulation, these cells have the potential to provide CYP function within a BAL.
|Title:||An investigation of Cytochrome P450 function in human cell Lines for potential use in a bioartificial liver device|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Medical Sciences > Medicine (Division of) > Internal Medicine|
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